Charles E. Hill Emory University School of Medicine

1. Converting BCR-ABL1 to the International Scale: Standardizing the Measurement of Relative Gene Expression • Charles E. Hill • Emory University School of Medicine

2. Outline • Measuring BCR-ABL1 • The International Scale • Approaches for calibrating to the IS • Challenges of relative gene expression testing

3. Disclosures • No financial interests/compensation from any of the companies discussed • Assay kits were provided for evaluation by both Asuragen and Ipsogen

4. Chronic Myelogenous Leukemia • 1.6 cases per 100,000 • 1.75:1 male:female • BCR-ABL1 translocation (Ph chromosome) • Molecular monitoring of BCR-ABL1 has become standard of care SEER data 2012

5. Why test? • BCR-ABL1 targeted by small molecules • Prognosis • Monitoring response • Prediction of relapse

6. Monitoring BCR-ABL1 • Karyotype • Fluorescence in-situ Hybridization • PCR • RT-PCR • Competitive RT-PCR • Nested RT-PCR • qRT-PCR

7. Sensitivities • Karyotype – 1:20 • FISH – 1-2:200 • RT-PCR – 1:100,000

8. Response to Therapy Hughes, NEJM, 2003, 349:1423

9. Prediction of Relapse Press, Clin Cancer Res, 2007, 13:6136

10. NCCN Guidelines • From NCCN 2.2013, qPCR should be performed: • At diagnosis • Every 3 months when responding to therapy. After CCyR, every 3 months for 3 years, then every 3-6 months • For rising transcript levels (1 log) with MMR, repeat in 1-3 months

11. The IRIS Trial • The International Randomized Study of Interferon versus STI571 • Major Molecular Response = 3 log decrease from average level at diagnosis • Three laboratories harmonized results (Australia, UK, and USA)

12. BCR-ABL1 in Practice • Many labs test for BCR-ABL1 • Testing and reporting are variable • Many report change from diagnostic (local) baseline, but not calibrated the same • Results not generally comparable between labs

13. Why is testing variable? • Sample volume and integrity • Processing and extraction • Reverse transcription • Control gene (BCR, ABL, GUSB, G6PDH, β2M) • Primers • Quantification standards • Instrument and analysis

14. Harmonizing Results • Median measurement of 30 shared baseline samples established as 100% • 3 log decrease from baseline (0.1%) is Major Molecular Response • For example, if median ratio BCR-ABL/BCR is 0.75, MMR = 0.00075 • International Scale defined by these two points

15. Getting to the IS • 3 IRIS trial labs use IS by definition (Adelaide, Seattle, London) • IRIS trial samples used to define 100% IS are not available to individual labs • Sample exchange with a laboratory calibrated to the IS?

16. Pre-WHO Standard • Branford, et al., Blood, 112:3330, 2008, “Desirable performance characteristics for BCR-ABL measurement on an international reporting scale to allow consistent interpretation of individual patient response and comparison of response rates between clinical trials,” • Exchange of patient samples with IS calibrated reference lab to determine Conversion Factor • Requires many samples and periodic re-assessment • Very burdensome for few reference labs

17. Calibration Pre-WHO Standard Branford, Blood, 2008, 112:3330

18. What does an IS do? • An International Scale helps standardize quantification • An IS does not improve pre-analytic issues • An IS does not reduce inherent assay variability • An IS does improve reporting

19. BCR-ABL1 WHO Standard • 1st WHO International Genetic Reference Panel for quantitation of BCR-ABL translocation by RQ-PCR • Intended for manufacturers/labs to develop secondary standards • 4 ampules of freeze dried cells • K562 cells (e14a2) diluted to different ratios in HL60 cells • Spanning relevant range of %IS values

20. WHO BCR-ABL1 Standard

21. Secondary Standards • Asuragen – ARQ IS Calibrators and BCR\ABL1 Quant • Ipsogen – BCR-ABL Mbcr-IS assay

22. Asuragen ARQ IS Calibrator Panel http://www.asuragen.com/Diagnostics/US/Products/qRT-PCR_Oncology/ARQ_IS_CAL/arq_is_cal.aspx

23. Asuragen Calibrators http://www.asuragen.com/Diagnostics/US/Products/qRT-PCR_Oncology/BCR_ABL1/

24. Ipsogen Calibrator • Single point calibrator • Tied to WHO Standard • Plasmids • Set to approximate 0.1% IS (MMR cutoff) • Specific for Ipsogen assay

25. Correlation of two IS based assays

26. Comparison to Predicate Test

27. Comparison to Predicate • Very similar results for both calibrated to the IS • IS tests are 0.4 and 0.35 lower than LDT • Very important to communicate this to clinicians when changing reports • Alternatively, report old result and IS

28. Advantages of Secondary Standards • Allow calibration to WHO standard • Simpler than exchanging many samples with reference lab • Manufacturer’s QC and lot-to-lot control • Periodic checks for drift are more feasible

29. Control Genes and Quantification • ABL1 is the most commonly used control gene • BCR was used as control gene for IRIS trial labs • GUSB used by some • EAC found ABL1, GUSB, and B2M suitable (Beillard, Leukemia, 2003, 17:2474) • BCR was not reported in study by EAC

30. ABL1 as Control

31. BCR as Control

32. Other genes as Controls • Do not participate in the translocation • Should be expressed constitutively and not vastly over/under expressed compared to transgene • GUSB and B2M

33. Limit of Detection • Depends on transgene copies and control gene level • What is minimum control gene for acceptable sensitivity? • Calculate minimum control gene level to detect MMR?

34. “Complete” Molecular Response CMR = no detectable BCR-ABL transcripts Press et al, Clin Cancer Res 13, 6136 (2007)

35. Practical LOD Issues • Balance sensitivity with not rejecting too many specimens • For LOD=5 copies BCR-ABL, need 50,000 copies of control gene to get 4.0 log • For 5.0 log need 500,000 copies control gene • LOD is affected by total RNA input (5 copies in isolation easier to detect than 5 copies with background nucleic acid)

36. Summary • The WHO Standard and development of IS have better standardized testing and reporting of BCR-ABL1 • Development of assays/calibrators tied to the IS make transition much simpler • Are more sensitive assays needed?

37. Thank You • Ruan Ramjit, Kaiser Permanente • Karen Mann, Emory • International BCR-ABL Standardization Group • Emmanuel Labourier, Asuragen • Mary Christopher, Ipsogen